The major aim of this project is to elucidate the role of sulfhydryl groups in the regulation of microtubule assembly in vivo and in vitro. We intend to purify two types of microtubule-associated protein--tau and HMW 2--and mix them with alkylated tubulin to see to what extent tubulin has to be alkylated in order for microtubule assembly to be blocked. Radioactive alkylating agents will be used in order to label the critical sulfhydryls which will be identified by peptide mapping and sequencing. Critical sulfhydryls in tau and HMW 2 will also be examined. The most effective alkylating agent we have used is ethylene-bis(iodoacetamide). Evidence from studies on assembly inhibition indicate that the critical sulfhydryls may be located on the lateral surface of the tubulin dimer, at or near the colchicine and podophyllotoxin binding site. We have also found a form of tubulin, apparently tissue-specific, with an altered beta-tubulin, that does not appear to react with ethylene-bis(iodoacetamide). The nature of this interaction will be elucidated. Vinblastine-induced aggregation of tubulin is much less sensitive to alkylating agents than is microtubule assembly.